Receptor tyrosine kinases and serine/threonine kinases have been implicated in cellular signaling pathways that control cell function, division, growth, differentiation, and apoptosis through reversible phosphorylation of the hydroxyl groups of tyrosine or serine and threonine residues, respectively, in proteins. In signal transduction, for example, extracellular signals are transduced via membrane receptor activation, with amplification and propagation using a complex choreography of cascades of protein phosphorylation, and protein dephosphorylation events to avoid uncontrolled signaling. These signaling pathways are highly regulated, often by complex and intermeshed kinase pathways where each kinase may itself be regulated by one or more other kinases and protein phosphatases. The biological importance of these finely tuned systems is such that a variety of cell proliferative disorders have been linked to defects in one or more of the various cell signaling pathways mediated by tyrosine or serine/threonine kinases.
Receptor tyrosine kinases (RTKs) catalyze phosphorylation of certain tyrosyl amino acid residues in various proteins, including themselves, which govern cell growth, proliferation and differentiation.
Downstream of the several RTKs lie several signaling pathways, including the Ras-Raf-MEK-ERK kinase pathway. It is currently understood that activation of Ras GTPase proteins in response to growth factors, hormones, cytokines, etc. stimulates phosphorylation and activation of Raf kinases. These kinases then phosphorylate and activate the intracellular protein kinases MEK1 and MEK2, which in turn phosphorylate and activate other protein kinases, ERK1 and 2. This signaling pathway, also known as the mitogen-activated protein kinase (MAPK) pathway or cytoplasmic cascade, mediates cellular responses to growth signals. The ultimate function of this is to link receptor activity at the cell membrane with modification of cytoplasmic or nuclear targets that govern cell proliferation, differentiation, and survival. Mutations in various Ras GTPases and the B-Raf kinase have been identified that can lead to sustained and constitutive activation of the MAPK pathway, ultimately resulting in increased cell division and survival. As a consequence of this, these mutations have been strongly linked with the establishment, development, and progression of a wide range of human cancers. The biological role of the Raf kinases, and specifically that of B-Raf, in signal transduction is described in Davies, H., et al., Nature (2002) 9:1-6; Garnett, M. J. & Marais, R., Cancer Cell (2004) 6:313-319; Zebisch, A. & Troppmair, J., Cell. Mol. Life. Sci. (2006) 63:1314-1330; Midgley, R. S. & Kerr, D. J., Crit. Rev. One/Hematol. (2002) 44:109-120; Smith, R. A., et al., Curr. Top. Med. Chem. (2006) 6:1071-1089; and Downward, J., Nat. Rev. Cancer (2003) 3:11-22.
The “Erk pathway” is an intracellular signal transduction pathway used by nearly all types of human cells to translate extracellular signals to cellular decisions, including proliferation, differentiation, senescence, or apoptosis (Wellbrock et al., Nat. Rev. Mol. Cell Biol. 11:875-885 (2004)). One of the invariant components of this pathway is the Ras GTPase, which receives signals from membrane receptors and activates the Raf protein kinases, which activate the Mek protein kinases, which in turn activate the Erk protein kinases. Activated Erk kinases phosphorylate a number of nuclear and cytoplasmic targets to initiate various cellular decisions. The biological importance of Raf in the Erk pathway is underscored by the finding that mutated forms of Raf are associated with certain human malignancies (see e.g. Monia et al., Nature Medicine 2:668-675 (1996); Davies et al., Nature 417:949-954 (2002)). Three distinct genes have been identified in mammals that encode Raf proteins; a-Raf, b-Raf and c-Raf (also known as Raf-1) and isoformic variants that result from differential splicing of mRNA are known (Chong et al., EMBO J. 20:3716-3727 (2001)). The Erk pathway is mutationally activated in a number of human cancers, most often by mutation of the Ras or b-Raf genes. Mutations in Ras and b-Raf genes generally occur in the same tumor types, including cancers of the colon, lung and pancreas and melanoma, but are usually mutually exclusive. This suggests that activation of either Ras or Raf is sufficient for pathway activation and cancer progression.
Naturally occurring mutations of the B-Raf kinase that activate MAPK pathway signaling have been found in a large percentage of human melanomas (Davies (2002) supra) and thyroid cancers (Cohen et al J. Nat. Cancer Inst. (2003) 95(8) 625-627 and Kimura et al Cancer Res. (2003) 63(7) 1454-1457), as well as at lower, but still significant, frequencies in the following: Barret's adenocarcinoma, billiary tract carcinomas, breast cancer, cervical cancer, cholangiocarcinoma, central nervous system tumors including primary CNS tumors such as glioblastomas, astrocytomas and ependymomas and secondary CNS tumors (i.e., metastases to the central nervous system of tumors originating outside of the central nervous system), colorectal cancer, including large intestinal colon carcinoma, gastric cancer, carcinoma of the head and neck including squamous cell carcinoma of the head and neck, hematologic cancers including leukemias, acute myelogenous leukemia (AML), myelodysplastic syndromes and chronic myelogenous leukemia; Hodgkin's lymphoma, non-Hodgkin's lymphoma, megakaryoblastic leukemia and multiple myeloma, hepatocellular carcinoma, lung cancer, including small cell lung cancer and non-small cell lung cancer, ovarian cancer, endometrial cancer, pancreatic cancer, pituitary adenoma, prostate cancer, renal cancer, sarcoma, and skin cancers.
By virtue of the role played by the Raf family kinases in these cancers and exploratory studies with a range of preclinical and therapeutic agents, including one selectively targeted to inhibition of B-Raf kinase activity (King A. J., et al., (2006) Cancer Res. 66:11100-11105), it is generally accepted that inhibitors of one or more Raf family kinases will be useful for the treatment of such cancers or other condition associated with Raf kinase.
Mutation of B-Raf has also been implicated in other conditions, including cardio-facio cutaneous syndrome (Rodriguez-Viciana et al Science (2006) 311(5765) 1287-1290) and polycystic kidney disease (Nagao et al Kidney Int. (2003) 63(2) 427-437). Since tumor cells frequently become dependent to one or two key signaling pathways for their survival (see, e.g. Jonkers et al., Cancer Cell. 6:535-538 (2004)), the Erk pathway represents a highly attractive target for drug intervention to treat cancer. Protein kinases in general are considered desirable targets for drug therapy, as evidenced by recent successes in targeting growth factor receptor and intracellular tyrosine kinases. Inhibitors of Mek have shown promise in clinical trials, however, there is ample evidence to indicate Mek-independent Raf signaling that may also contribute to cancer progression (Wellbrock et al, Nat. Rev. Mol. Cell Biol. 11:875-885 (2004)). Therefore, targeting Raf kinases promises an alternative and complementary approach to treating tumors in which Ras or Raf genes are mutated.